Respiratory infection is the most common lung disease with an estimated incidence of over one billion cases annually in the United States. Gram negative bacteria, particular Pseudomonas aeruginosa, are opportunistic pathogens that frequently cause hospital-acquired infections especially in ventilated patients and are the second most common infection in Intensive Care Units. The conducting airway serves as a first line of defense against bacterial insults through passive action as a barrier as well as through active contribution to antimicrobial activity of the extracellular lining fluid. Importantly, airway epithelial cell-derived proteins contribute to this milieu and promote an appropriate host defense against invading pathogens. Our long-term objective is to advance the understanding of the respiratory innate immune response especially with regard to epithelial cell-specific antimicrobial function. One molecule we believe to be critical to epithelially initiated antimicrobial defense is PLUNC (palate, lung and nasal epithelium carcinoma associated) which we co-discovered and originally named SPURT (secretory protein in upper respiratory tract). We characterized PLUNC as airway lining fluid protein that was secreted apically in primary human airway epithelial cultures and whose abundance was elevated in multiple chronic lung diseases. We demonstrated that PLUNC mRNA and protein in normal subjects were restricted to serous cells of secretory ducts and submucosal glands, sites that express high levels of other host defense proteins such as lysozyme and lactoferrin. PLUNC is abundant in airway fluid of normal individuals and our preliminary results demonstrate that it possess antimicrobial activity against P. aeruginosa. In this study, we hypothesize that mucosal PLUNC acts as an antimicrobial protein that is a critical determinant in host defense against Gram-negative bacteria. To test this hypothesis, we will pursue three specific aims. For Aim 1, the specificity of PLUNC toward common airway pathogens will be assessed and necessity of PLUNC for anti-microbial activity evaluated in vivo and in vitro. For Aim 2, PLUNC transgenic mice and prophylactic administration of recombinant PLUNC will be used to determine if elevated epithelial PLUNC protects against respiratory microbial infection. For Aim 3, the peptide sequences and protein motifs of PLUNC critical to its antimicrobial function will be mapped and utility or these peptides as endogenously generated antimicrobial agents tested. PUBLIC HEALTH RELEVANCE. Relevance to Public Health Concerns: These studies will assess innate mechanisms regulating antimicrobial activity of the airway lining fluid. By focusing on an epithelially-derived protein product, PLUNC, our study may provide an alternative treatment for respiratory infection through augmentation of native host defense mechanisms.